Glucosamine, the intermediate metabolite of the hexosamine biosynthesis pathway (HBP), is widely used as a supplementary drug in patients with osteoarthritis. However, its consequences in such patients concomitantly suffering from diabetic nephropathy is unknown.in vivo and cultured endothelial cells in vitro were used in the study. The mice were treated with glucosamine for 6 months. Renal function was evaluated by metabolic cage, and histology of the kidney was estimated by periodic acid-schiff (PAS) staining. The expression of related genes was assessed by real-time PCR, immunofluorescence staining, immunoblotting and ELISA.The aim of the study was to investigate the effect of exogenous administration of glucosamine in the diabetic kidney. A mouse model of streptozotocin-induced diabetic nephropathy There was no significant difference in urinary albumin secretion, relative kidney weight, or creatinine clearance between the groups treated with glucosamine and controls. Assessment of the kidney demonstrated reduction in mesangial expansion and fibronectin expression in the diabetic glomeruli treated with glucosamine. Glucosamine treatment significantly decreased α-smooth muscle actin (α-SMA) protein expression in both diabetic and control kidneys, whereas the expression of other fibrosis-related genes and inflammatory factors was unaltered. Moreover, α-SMA colocalized with the endothelial marker CD31 in the diabetic and control kidneys, and glucosamine reduced α-SMA+ ECs in the diabetic glomeruli. In addition, glucosamine suppressed α-SMA expression in endothelial cells treated with or without high glucose.In summary, this is the first report to show that glucosamine reduces mesangial expansion and inhibits endothelial-mesenchymal transition in diabetic nephropathy. The underlying mechanisms need to be further investigated. Diabetic nephropathy (DN) is a common chronic complication of diabetes mellitus, affecting around 30 and 40% of patients with type 1 and 2 diabetes mellitus, respectively , 6. TGF-Glucosamine is a physiologically occurring sugar molecule in the HBP and an essential component of glycoproteins, proteoglycans, and glucosaminoglycans . It is fin vitro was also demonstrated in that study  Karlsruhe Regierungspräsidium and carried out in C57Bl/6 male mice. The animals had free access to food and water and were cared by trained staff. The animals were randomly divided into 4 groups, NC: normal control group: NC+G: normal mice treated with glucosamine; DC: diabetic control group; DC+G: diabetic mice treated with glucosamine. Diabetes was induced by intraperitoneal injection of streptozotocin  into approximately 10-week-old mice. A successful induction of diabetes was defined when blood glucose was more than 250 mg/dl 1 week after STZ injection. The glucosamine was mixed into the standard food  which was supplied to the control mice. The mixture of the glucosamine was performed in the company SSNIFF. Oral glucosamine treatment via food  wasAt the end of the experiments, the mice were weighed in the late afternoon and placed in metabolic cages for 16 h to assess the general metabolic parameters and renal function. The mice had free access to food and water in the cages. In the metabolic cages, food and water intake, urinary excretion, and feces weight were determined. Additionally, serum was collected and stored at –80°C for further analysis. Measurement of urinary albumin, creatinine, and urea was performed on a Hitachi 917 Autoanalyser . Simultaneously, levels of blood glucose, HbA1c, creatinine and urea were measured in the plasma.Periodic acid-schiff (PAS) staining was used to evaluate the histology and mesangial expansion in the renal cortex. Paraffin sections of kidney (4 μm) were dewaxed at 60°C for 1 h and deparaffinized in Roti-Histol and hydrated in a descending ethanol series. Then, the sections were place in 1% periodic acid for 10 min and Schiff’s reagent for 10 min followed by a staining with Mayer’s Hematoxylin for 1 min. After washing in an increasing ethanol series and incubation in Roti-Histol, the sections were embedded with Entellan. Samples were observed with a Leica microscope and images were taken with 40x objective, and analyzed using ImageJ. The mesangial expansion area  was calculated as the proportion of the area of the mesangial matrix to the total area of the glomerulus and presented in percentage as described by Rangan and Tesch .Paraffin sections were used for the immunofluorescence assessment of α-SMA and CD31. Following a similar deparaffinization and hydration procedure as previously described, the antigens were unmasked using a citrate retrieval buffer. Cryosections were used for the staining of fibronectin and fixed with acetone. Unspecific binding was blocked with a 1% BSA blocking buffer and the cell membranes were permeabilized with 0.5% Triton-X 100 for 1 h at room temperature. The sections were further incubated with the primary antibody mouse anti-α-SMA (1:500 Sigma A5228) and endothelial marker CD31 (1:200 Santa Cruz sc-1506-R) for colocalization, or rabbit anti-mouse fibronectin (1:200 Sigma F3648) overnight at 4°C. After washing on the second day, samples were incubated with the corresponding goat anti-mouse secondary antibody labeled with Alexa Fluor 488  and/or swine anti-rabbit TRITC  for 1.5 h, then counterstained with DAPI. Finally, the sections were covered with Roti FluorCare mounting medium and images were taken using a confocal microscope . The quantification of the immunofluorescence staining in the samples was done according to the protocols described in Rangan and Tesch . BrieflyHuman umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords with agreement of patients according to a previously described protocol. The use of HUVECs was approved by the local medical ethics committee . HUVECs were cultured in ECBM containing 10% FCS in a 1% gelatin-coated cell culture dish. HUVECs until passage 4 were used for the experiments. 24 h after seeding cells for the experiments, HUVECs were serum-starved in ECGM with 0.5% FCS for 24 h followed by stimulation with high glucose (30 mM D-glucose) with or without glucosamine in 0.5% FCS for additional 24 h.Proteins from the renal cortex or HUVECs were extracted in RIPA buffer with proteinase inhibitors, and protein concentrations were determined using the BCA assay. Twenty-five microgram of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with Roti-block  for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C and corresponding secondary antibodies for 1 h at room temperature. The proteins were visualized using a chemiluminescent peroxidase substrate . Protein expression was quantified using Image J . Specific primary antibodies used: mouse-anti-α-SMA , goat-anti-CTGF , mouse-anti-γ-tubulin . The secondary antibodies conjugated with horseradish peroxidase: rabbit-anti-goat , horse-anti-mouse .The proteins dissolved in RIPA buffer were used for detecting IL-1β levels in the kidney cortex. For this experiment, the IL-1β ELISA kit for sandwich ELISA from R&D Systems (MLB00C) was employed according to the manufacturer’s instructions. In brief, proteins were pipetted into a 96-well plate coated with the IL-1β antibody. After incubation for 2 h at room temperature, the plate was washed to remove unbound proteins. The secondary antibody coupled with enzyme was pipetted into the wells and incubated for 2 h at room temperature. Finally, the substrate was added into the wells and the reaction was stopped by a stop solution. The plate was read with a photometer and the data was evaluated.Real-Time Quantitative PCR were carried out as described previously . In briePost hoc intergroup was compared with the Tukey correction. GraphPad Prism 5  was utilized for the statistical analysis. p values < 0.05 were considered significant.The data are presented as mean ± SEM. The results were compared using an analysis of variance. p < 0.001). Treatment with glucosamine led to a significant (21%) reduction in the formation of the mesangial matrix in diabetic mice (p < 0.01). In the control mice, there was no significant change between animals with and without glucosamine treatment. Furthermore, we estimated the expression of fibronectin and collagen 4 using immunofluorescence staining. Fibronectin expression was significantly promoted by hyperglycemia. Glucosamine significantly reduced the expression of fibronectin elevated by hyperglycemia , suggesting hypertrophic alteration in the diabetic kidney. Nevertheless, treatment with glucosamine did not significantly change the ratio in either NC or DC animals.Firstly, we investigated the effect of glucosamine on kidney function by assessment of kidney weight and ratio of kidney weight to body weight between the groups . The kidp < 0.001). Supplementation of glucosamine did not change albumin excretion in either diabetic or non-diabetic mice. No changes in ACR upon glucosamine treatment were detected . Nevertheless, glucosamine showed no effect on the RNA expression of FN-1 in the kidney . Glucosamine treatment showed no effect on its expression levels in the diabetic as well as in the non-diabetic kidneys. To verify the observation in IL-1β mRNA expression, we also assessed the IL-1β expression by ELISA using the cortex lysates, detecting a similar expression pattern with even a trend toward protein downregulation with hyperglycemia and glucosamine treatment.Another aspect of diabetic nephropathy is the immigration of inflammatory cells and the release of inflammatory proteins. According to the feature of glucosamine in inhibiting inflammatory factors, the effect of glucosamine on inflammatory factors such as IL-1ß and TNF-α was also investigated by quantitative real time PCR (data not shown). The mRNA expression of TNF-α in diabetic kidneys tended to be elevated, however, did not reach significance compared to the control kidneys. Its expression was comparable in the groups with or without glucosamine treatment. Unexpectedly, IL-1β mRNA expression in the diabetic cortex tended to be lower than that in the NC group (p < 0.001), while it was moderately expressed in the control kidney. Glucosamine therapy significantly (27%) decreased α-SMA expression in both diabetic (p < 0.01) as well as in non-diabetic mice (p < 0.05).Based on the fact that glucosamine reduced the accumulation of extracellular matrix and expression of fibronectin in the glomeruli, but the transcriptional gene expression of extracellular components and inflammatory factors were not changed upon glucosamine treatment, we examined the protein expression of different markers for renal fibrosis such as fibronectin, collagen, connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) using immunoblotting . The wesTo confirm this result from the western blot and to know whether α-SMA is regulated in glomeruli as well, immunofluorescence staining of the kidneys with α-SMA antibodies was performed . A signiUpon discovering the regulation of α-SMA expression by glucosamine in the glomeruli, next, we assessed the cell-specificity. We performed experiments colocalizing α-SMA with podocyte marker Nephrin. However, no obvious colocalization of α-SMA and nephrin was identified (data not shown). We also performed immunofluorescence staining using α-SMA and a cell marker of endothelial cells (CD31) . Unexpecin vivo findings, we evaluated whether glucosamine modulates the expression of α-SMA in endothelial cells in vitro. HUVECs were stimulated with and without high glucose and treated with glucosamine. In accordance with the observation showing α-SMA+ endothelial cells in the control glomeruli in vivo, the immunoblotting results demonstrated that α-SMA could be detected in the control cells cultured in normal glucose , 2012-338N-MA. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Regierungspräsidium Karlsruhe, G178/15.YF designed the study. LT, RE, UT, JW, FS, ML, AA, and YW performed the experiments. LT, RE, H-PH, and YF interpreted the data. LT, RE, and YF wrote the manuscript with input from all authors. H-PH and YF provided essential expertise and knowledge of metabolic diseases for the study. All authors have approved the manuscript for publication.


To date, the impact of these variables has not been assessed in the setting of HIV-associated DLBCL (HIV-DLBCL), which is among the most common malignancies seen in people living with HIV. In this study, we assessed these factors in a cohort of South African patients with DLBCL and a high HIV-seropositivity-rate. In addition, we evaluated the prognostic value of monocyte activation (as reflected by monocyte fluorescence (MO-Y) on a Sysmex haematology analyser). This parameter has to date not been assessed in the setting of DLBCL.Diffuse large B-cell lymphoma (DLBCL) is a high grade non-Hodgkin lymphoma which is common among immunodeficient people. Derangements of peripheral blood immune cells have been described to have a prognostic impact in DLBCL in high income countries, including a monocytosis, the ratios of lymphocytes to both monocytes (L:M) and neutrophils (N:L), as well as the numbers of regulatory T-cells (Tregs) and immunosuppressive monocytes , the N:L (>6:1), high numbers of Tregs (≥5.17% of CD4s) and lymphopenia (<1.3 x 109/L) were. In addition, increased monocyte fluorescence (MO-Y >115.5) was associated with superior outcomes, which we speculate to reflect a more robust antitumour immune response among individuals with high levels of monocyte activation. On Cox Proportional hazard analysis, immune-cell factors independently associated with survival included a CD4 count <150 cells/ul and a neutrophilia.Seventy-six patients were included, of whom 81.3% were people living with HIV with a median CD4 count of 148 cells/ul. Most patients had advanced stage disease (74.8%) and were predominantly treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based chemotherapy (without Rituximab). At a median follow-up period of 19 months, the median survival time was 3.5 months, with a 12-month survival rate of 27.0%. All of the immune-cell-related variables (with the exception of the CD4 count) were similar between the people living with HIV and the HIV-negative individuals. In contrast to previous studies, a high monocyte count, the L:M and increased numbers of HLA-DRlow monos are not strong prognostic indicators in HIV-DLBCL, while a low CD4 count and neutrophilia are. Elevation of the MO-Y shows some promise as a potential biomarker of antitumour immunity; further study in this regard would be of interest.The monocyte count, L:M and the number of HLA-DR The immune system plays a key role in preventing the evolution of cancer through the recognition and elimination of malignant cells, as well as through clearance of oncogenic viruses. This is evidenced by the increased predisposition to cancer seen among people living with HIV infection and other immunodeficiencies (such as inborn errors of immunity), where chronic immune stimulation due to recurrent or persistent infections predisposes particularly to malignant lymphoid proliferations (such as non-Hodgkin lymphoma (NHL)).low monos) [low monos form part of the homeostatic response to immune activation, functioning to downregulate the immune response and prevent immune-mediated tissue damage. To date, the impact of the monocyte count, the L:M, the N:L and the number of both Tregs and HLA-DRlow monos have not been assessed in the setting of HIV-associated DLBCL (HIV-DLBCL). In this study, we aimed to assess these factors in a cohort of South African patients with DLBCL with a high HIV-seropositivity rate. In addition, we evaluated the prognostic value of monocyte (MO-Y) and neutrophil (NE-SFL) fluorescence, which have to date not been assessed in the setting of DLBCL. These parameters are measured with the routine differential count on Sysmex XN-series haematology analysers , and represent a cost-effective marker of monocyte and neutrophil activation, respectively.Monocytes and neutrophils are peripheral blood immune cells which play a key role in the innate immune response. Monocytes are produced in the bone marrow, and circulate for 1–3 days in the peripheral blood before egressing into tissues where they give rise to macrophages and dendritic cells. Monocytes play a role in both normal and malignant B- and T-cell function, and can support proliferation of malignant lymphoid cells . Neutropw monos)   with incident DLBCL diagnosed at or referred to Chris Hani Baragwanath Academic Hospital (CHBAH) in Soweto, Johannesburg for specialist management between November 2019 and May 2022. CHBAH is a large tertiary state-sector hospital which provides specialist haematology-oncology services to a large population with poor socio-economic status in the Southern Gauteng province (including the south of the city of Johannesburg), as well as the North West Province of South Africa. All patients referred over this period with an available biopsy of sufficient quality for further immunoshistochemical testing were included. When possible, additional testing was performed on peripheral blood samples, including assessment of the MO-Y, NE-SFL and enumeration of Tregs and HLA-DRlow monocyte numbers were determined by Receiver Operator Curve (ROC) analysis, with the values demonstrating the best balance between test sensitivity and specificity for predicting patient survival (as evidenced by a high likelihood ratio (LR) with both high sensitivity and high specificity) being selected. Univariate survival analysis was performed using Kaplan-Meier survival estimates, and median survival times were compared using log rank tests. This was performed on the entire cohort as well as among the people living with HIV . Unfortunately, this sub-analysis could not be meaningfully performed in the HIV negative patients due to the small size of this group. Patients who were clinically stable but lost to follow-up before they were referred to the Clinical Haematology Unit were excluded from the survival analysis, while those who were lost to follow-up after having commenced treatment were included. The one-year overall survival (OS) rate was calculated as the proportion of patients who were still alive 12 months after diagnosis. Living patients who had been followed up for <12 months were excluded from this analysis. Cox proportional-hazards models were used to investigate the association between the survival time at the time of write-up and predictor variables which showed a statistically significant association with survival on Kaplan-Meier analysis. As with univariate analysis, this was performed both on the whole cohort and specifically among the people living with HIV. The assumptions of proportionality were confirmed to have been met by means of Schoenfield analysis of proportionality. Statistical analysis was performed using Prism software, version 5 , at https://statpages.info/prophaz.html  and at https://acetabulum.dk/cgi-bin/cox . Statistical significance was accepted at a two-sided p-value of 0.05.Continuous data are presented as the median (interquartile range (IQR)) and categorical data as frequencies and percentages. The Mann-Whitney U-test and Fishers exact test were used to compare continuous and ordinal variables of interest respectively, and correlation between variables of interest was assessed using Spearman’s correlation. Correlations were performed on all the results pooled, irrespective of HIV-status. Owing to limited numbers, cases with low (0–1), intermediate (2–3) and high IPI scores (4–5) were pooled for analysis and referred to as the IPI class. Optimal cut-off values for the lymphocyte count, monocyte count, neutrophil count, L:M, N:L, MO-Y, Treg and HLA-DRThe study included 76 patients with a median age of 42 years and a male to female ratio of 1.3:1. HIV test results were available in 75/76 of the patients, of whom 61 (81.3%) were people living with HIV. Information about ART exposure was available in 54 patients, 44 of whom (81.5%) were on ART at the time of referral. However, of these, 13 (29.5%) had been commenced on ART after the onset of their symptoms of lymphoma. The CD4 count was <150 cells/ul in 33 (54.1%) of the people living with HIV, of whom 85% were on ART for a median of 6.5 months (IQR 2–45), and 21.4% were virologically suppressed (HIVVL <100 copies/ml). Among patients with higher CD4 counts (>150 cells/ul), virological suppression (VS) was significantly more common , and the duration of ART exposure was significantly longer (24.5 months (IQR 14.3–63); p = 0.048). Relevant clinical and histological data are summarized in 9/L) was present in 29/76 (38.2%) of the patients, and was not significantly more common in the people living with HIV ((26/61 (42.69%) vs 3/14 (21.4%) in the HIV negative group, p = 0.22). Monocytosis (>0.8 x 109/L) was present in 18/76 (23.7%) of the patients, and did not differ in frequency according to HIV-status (p = 1). There was a significant positive correlation between the CD4 count and the lymphocyte count (rs 0.56 (0.36–0.72); p<0.0001). On survival analysis, lymphopenia (<1.3 x 109/L) and a CD4 count <150 cells/ul were both significantly associated with a shorter survival time in the people living with HIV (9/L) was not.  was present in only 2/76 (2.6%) of the patients, both of whom were HIV negative. Neutrophilia (>7 x 109/L) was present in 22/76 (28.9%), and did not differ in frequency according to HIV-status (p = 1). There was a significant positive correlation between the neutrophil count and the monocyte count (rs 0.5 (0.31–0.66); p = <0.0001), but no significant relationship was seen between the neutrophil count and the lymphocyte count, the CD4 count, or the NE-SFL. On survival analysis, a neutrophil count above 8 x 109/L and a N:L ratio ≥6:1 were both significantly associated with a shorter survival time in the people living with HIV  were people living with HIV. The median HLA-DRlow monocyte count was 26.2%, and did not differ significantly according to HIV-status (low monocyte numbers and the absolute monocyte count (rs 0.5 (0.2–0.71) p = 0.001) and the absolute neutrophil count (rs 0.61 (0.36–0.78); p<0.0001). There was no significant correlation between the number of HLA-DRlow monocytes and the disease stage or the CD4 count. On survival analysis, survival times appeared substantially longer in patients with HLA-DRlow monocyte counts below 20%, but this finding lacked statistical significance, possibly due to the relatively limited number of cases with HLA-DRlow monocyte counts in this range (n = 11) (HLA-DRV-status . There w(n = 11) .Treg numbers were assessed in 29 patients not yet on corticosteroids, with an HIV-seropositivity rate of 79.3%. The median Treg count as a proportion of CD4 T-cells was 5.62% (IQR 1.65–9.27), with a median absolute Treg count of 7.45 cells/ul (IQR 2.33–17.8). Treg counts did not differ significantly according to HIV-status , but thes -0.66 (-0.85 to -0.32) p = 0.0008) and CD39 expression (rs -0.47 (-0.72 to -0.12) p = 0.01) with the CD4 count. On survival analysis in the global cohort, patients with a Treg count >5.17% (as a proportion of CD4 cells) and Treg expression of CD39>93% had significantly shorter survival ; p = 0.04). In addition, it showed marginally significant negative relationships to the monocyte count (rs -0.25(-0.49 to 0.02); p = 0.06), the HLA-DRlow monocyte count  and the neutrophil count . The NE-SFL was measured in 65 patients and was similar between the people living with HIV and the HIV negative group. On survival analysis, survival times were significantly shorter in patients with MO-Y levels below 115.5, and were marginally longer in patients with a NE-SFL <50.5  were people living with HIV. The median MO-Y value was 110.9, and did not differ according to HIV-status . It showFL <50.5 . Among p9/L  has been shown to be a significant predictor of survival on gene expression profiling studies . PeripheIn contrast to reports from other parts of the world , 7, 11, 9/L) and a low CD4 count with survival on multivariate analysis. Interestingly, neutrophils were shown by Gregoire at al3 to support tumour cell growth and survival through production of BAFF/APRIL, and to induce changes in the stromal components of the TME which favour the support of malignant B-cells. At the same time, the stromal cells are induced to produce interleukin 8 and C-C motif ligand 2 (CCL2), thereby recruiting more neutrophils and monocytes to the TME, respectively. These dynamics within the TME may account for the neutrophilia and monocytosis seen in 28.9% and 23.7% of our patients, respectively, and may contribute to the poor survival rates seen in patients with a neutrophil count >8 x 109/L.As has been reported previously –21, we flow) monocyte numbers were high (>25%) in this cohort, and did not differ significantly according to HIV-status. The findings are in agreement with those from a previous study performed in the USA,11 which showed an average of 35.2% HLA-DRlow11 monocytes in patients with advanced stage DLBCL. However, in contrast to this report, we found no correlation between the number of HLA-DRlow monocytes and disease stage, and while survival rates appeared superior in patients living with HIV with HLA-DRlow mono counts <20%, this finding was not statistically significant. Notably, the HLA-DRlow monocyte count showed significant correlations with both the absolute monocyte and neutrophil counts, likely reflecting the effects of a sustained pro-inflammatory stimulus which drives both myeloid cell proliferation and induces homeostatic downregulation of the immune response. In addition, there was a marginally significant inverse correlation between the number of HLA-DRlow monocytes and monocyte fluorescence (MO-Y) levels, which were significantly associated with inferior survival on univariate survival analysis (possibly due to their relationship with a poorer PS). We have previously shown MO-Y levels to be correlated to monocyte CD64 expression, thus suggesting that this parameter reflects monocyte activation [low monocytes was mediated by arginine metabolism, and that arginine supplementation partially reversed this phenotype [Immunosuppressive  did. We speculate that this finding could reflect a more robust antitumour immune response among individuals with elevation of the MO-Y. As an easily measured, inexpensive and widely available parameter, the MO-Y holds appeal as a potential biomarker of antitumour immunity. Further study in this regard would be of interest.The peripheral blood monocyte count and L:M are not strong prognostic indicators in HIV-DLBCL, and while a low lymphocyte count is associated with poorer survival, this is a product of low CD4 counts, and not lymphopenia per se. In contrast, a neutrophil count >8 x 10An important limitation to this study is the small number of patients who were HIV negative. As such, survival analysis of the HIV negative group was not possible. Additional limitations include the incomplete nature of the available data in a subset of the patients, and the limited exposure to Rituximab therapy in this cohort. Lastly, comparison of Treg numbers between the people living with HIV and the HIV-negative group was not adjusted for age and sex, which may have had an impact on these results.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


The glucagon-like peptide 1 (GLP-1) is a hormone which is produced in the enteroendocrine L-cells in the ileum and the neurons of nucleus tractus solitarius (NTS) in the brain which has numerous metabolic effects. The central GLP-1R's role in cognitive functioning is well known. On the contrary, it has been shown that exercise has positive effects on brain function. So, we decided to elucidate whether the central GLP-1 has a role in memory and learning. Thirty-two rats were used in this experiment in 4 groups. After anesthetizing the rats, the right lateral ventricle was detected, and a cannula was directed to the ventricle. Ten micrograms of exendin-3 or sterile saline, according to the group, was injected via ICV once daily for seven days. The rats in the exercise group considered an exercise period of one hour each day (17 meters per minute) for seven consecutive days. To evaluate the performance of memory and learning, a standard Morris water maze (MWM) tank was utilized. According to the results, the TE-exendin group showed a statistically significant difference from the TE-SAL group in both parameters of latency and time in the zone. In summary, memory and learning were improved by GLP-1R in the exercise group, but not in the sedentary group, which we can hypothesize that exercise can affect memory and learning through this pathway. Memory and learning loss is regarded as one of the world's greatest issues brought on by age, accidents, and even some pharmaceuticals. Although several research have been conducted on medicines to prevent age-related cognitive decline, effective treatments for cognition and memory enhancement are not yet available . The glThe central GLP-1R in cognitive functions is involved, and it is studied . SeveraIt is well accepted that physical activity has beneficial effects on brain activities . HoweveEvidence suggests that exercise increases the GLP-1 levels . It wasBased on these investigations, we decided to elucidate whether the central GLP-1 receptors have a role in the beneficial effects of exercise on L&M. Simultaneously, we revalidate the previously reported positive effects of GLP-1 on L&M performance.μg/rat, 10 μl)  was mixed in normal saline and injected in intracerebroventricular (ICV) manner .Thirty-two male Wistar rats  were obtained from the Teb Azma animal institute for biological sciences. All animals were maintained in a 12 h light/dark cycle at 21–25°C. Rats were individually housed in standard polycarbonate cages with standard sawdust as bedding. Animals were fed a standard pellet diet which had ad libitum access to water. The current research was authorized by the Lorestan University Ethics Committee (LU. ECRA.2021.7) and conducted in accordance with the National Institutes of Health Guide for the care and use of laboratory animals. Exendin-3 (9-39) , and rats were placed on a stereotaxic apparatus . The method described by Paxinos and Watson (1987) was followed, and a stainless steel guide cannula gauge 23) was cautiously placed in the right lateral ventricle . To famn = 8):Sedentary saline (SED-SAL) groupSedentary-exendin-3 (SED-exendin) group3) Treadmill exercise-saline (TE-SAL) group4) Treadmill exercise-exendin-3 (TE-exendin) groupAll animals were randomly divided into four groups , daily for seven-day running period. After the last day of running, the animals were tested for L&M tests using Morris water maze (MWM) task.All rats received the injections just before the exercise manually by hand at a flow rate of 10 We used a standard MWM task which was given in previous reports . MWM prP < 0.5 is considered a statistically significant difference.The statistical analysis was performed using SPSS software for Windows . To examine the interaction among groups and days in 4 levels and to ascertain the significant effects (“group” effect and “day” effect) on escape latency, a two-way ANOVA with repeated measures (days) was utilized for data in the learning phase. Moreover, data regarding memory retention test were analyzed using a two-way ANOVA to specify the significant main effects (“group” effect and “zone” effect) on time spent percentage and the interaction among groups in 4 levels and zones in 2 levels of the target and opposite zones. All analyses were followed by a post hoc Tukey's test regarding the multiple comparison evaluation. All data were expressed as means ± SEM. F3,432 = 1.707, P < 0.01) and days  was revealed on the escape latencies. The results revealed that data regarding escape latencies of treadmill exercise-saline group were remarkably lower than that of sedentary saline group on the third and fourth days of the experiment. The TE-exendin group had a significantly longer escape latency than the TE-SAL group on the second, third, and fourth day (P < 0.05). The result of the memory retention test is shown in P < 0.05). As shown in P < 0.05).Acquisition data analysis of all groups is shown in In rats, moderate treadmill activity improves L&M performance via the MWM task, and ICV injection of exendin-3 blocks these favorable effects of exercise (9-39). These findings indicate that central GLP-1 receptors may mediate the effects of exercise on L&M. This is the first study we are aware of that has shown that exercise may exert its beneficial effects on cognitive functions via central GLP-1 receptors. This finding that exercise improves L&M confirms our previous study and other research indicating the beneficial effects of exercise on L&M  rats. Since ICV injection of exendin-3 had not shown adverse effects on L&M in normal situations and its use in exercised rats impaired the beneficial effects of exercise on L&M, it is likely to hypothesize that the valuable effects of exercise on L&M may be triggered by the central GLP-1R.Many reports hypothesized that exercise leads to improve brain function via regulating the GLP-1 release. It is shown that mild and moderate exercise increases GLP-1 concentrations in plasma. For instance, Ueda et al. indicated that an aerobic exercise protocol significantly increased GLP-1 concentration in blood . Its phSeveral mechanisms seem to play a role in exercise-induced enhancement of L&M via GLP-1 receptors. One possibility is an interaction between GLP-1 and hippocampal LTP. Presently, much evidence demonstrates that synaptic plasticity, including LTP, mediates hippocampal-dependent memory . On theβ (glycogen synthase kinase-3β), NFκB (nuclear factor-κB), and CREB (cyclic adenosine monophosphate response element-binding protein). Of note, CREB is a crucial player in modulating BDNF production. BDNF was widely studied in cognition, inflammation, and neurogenerative disorders. GLP-1 analogs, such as exenatide and liraglutide, have neuroprotective effects in animal models of Alzheimer's disease (AD) . The roFoltynie ).In general, we concluded that exercise-induced enhancement of spatial L&M function in rats was prevented by blockade of central GLP-1R using exendin-3. This result indicates that GLP-1 has a significant role to mediate the beneficial effects of physical exercise on cognitive functions.


Good sit-to-stand (STS) performance is an important factor in maintaining functional independence. This study investigated whether free-living STS transition volume and intensity, assessed by a thigh-worn accelerometer, is associated with characteristics related to functional independence.n = 287, men n = 192). The records were used to evaluate the number and intensity (angular velocity of the STS phase) of STS transitions. Associations with short physical performance battery (SPPB), 5-times-sit-to-stand test (5×STS), isometric knee extension force, self-reported fear of falls, and self-reported difficulty in negotiating stairs were also assessed.Free-living thigh-worn accelerometry was recorded continuously for 3–7 days in a population-based sample of 75-, 80-, and 85-year-old community-dwelling people . All variables were higher in men than in women (p < .001) and were positively associated with SPPB total points, knee extension force  and negatively associated with 5×STS , lower extremity functional limitations (p < .01), fear of falls (p < .01), and stair negotiation difficulties (p < .01).The number of STS transitions, mean and maximal angular velocity were lower in older age groups (Free-living STS characteristics were related to lower-extremity performance, lower extremity functional limitations, self-reported fear of falls, and stair negotiation difficulties, which can be a sensitive indicator of impending functional decline. Moreover, STS transitions may provide an indicator of adequacy of lower-limb muscle strength among older individuals. Sit-to-stand (STS) transitions are one of the most common activities of daily life  and goodThe recent miniaturization of technology has made prolonged recordings of free-living physical behavior feasible . InexpenPickford et al. 2019) have evaluated STS transition kinematics in the free-living environment using a proprietary algorithm to compare peak velocities of STS transitions between stroke survivors and unaffected peers , which was conducted in the Gerontology Research Center, the University of Jyväskylä. AGNES comprises three age cohorts  of people living independently in the city of Jyväskylä, in Central Finland. The study protocol has been published by Rantanen et al.  were used to develop algorithms to detect and quantify free-living STS transitions (n = 479), which was used to identify the volume and quantify the intensity of free-living STS transitions. The flow chart of the study has been reported elsewhere . In the first phase, the magnitude (Euclidian norm) of the resultant acceleration for each sampling instant was calculated from raw accelerometer data. Mean amplitude deviation (MAD) was calculated in nonoverlapping 5 s epochs based on the magnitude of the resultant acceleration .To identify the instantaneous orientation of the thigh, we calculated an angle for postural estimation (APE) from resultant acceleration values using the method described by Vähä-Ypyä et al. . The calSTS transitions were thereafter identified as follows: all posture estimation signal transitions from 0  to 1 (upright) were considered as candidate STS transitions. A candidate was accepted as a STS transition if the following 3 criteria were met: (a) the variance of the magnitude of the resultant acceleration between 2.5 s and 0.5 s prior to the candidate transition was less than 0.02 g , (b) starting angle of the STS transition  was more than 65 degrees (1.14 rad) and, (c) the movement of the transition ended at an angle of less than 35 degrees (0.61 rad). Due to the variance criterion the algorithm will only detect the first of a set of STS movements .The intensity of an identified STS transition was estimated based on the APE-signal time derivative  as follows: baseline APE (corresponds to thigh angle prior to standing up) was established as the mean between 2.5 s and 1.5 s to the identified transition instant. The last sample at the baseline value prior to the transition instant was thereafter set as the initiation of the angular velocity determination. Linear fits were then applied to each data set from the initiation sample until the transition instant to transition instant + 0.15 s with one sample length increment. The longest fit where the square of the last instant of the fit and the APE differed by less than experimentally determined 0.005 degrees was chosen, and the slope of the chosen fit is reported as the STS transition intensity. The STS transitions detection accuracy of the algorithm was examined using the laboratory session of this study. Prior to the 6-minute walk test, the protocol included two known STS transitions that were defined as the ground truth. Ground truth STS transitions identified by the algorithm were defined as true positives. Ground truth STS transitions that could not be identified were defined as false negatives. No false positives were identified, and we did not attempt to define a true negative. True positives and false negatives were used to calculate detection accuracy, which ranged between 82.7% and 97.5% depending on the age (better accuracy among younger age groups compared to older age groups) and sex (better accuracy among men than women) groups, with an overall accuracy of 93.3% . The angThe volume of the STS transitions was determined as the number of transitions per monitoring day, and the STS transitions mean intensity (mean median angular velocity) was determined as the mean of daily median transitions. The maximal intensity  was defined as the median of the ten fastest STS transitions over the entire monitoring period. No participant exceeded 4 rad/s in the laboratory, and therefore we filtered out any STS transitions above 4 rad/s from the data prior to the estimation of the maximum free-living angular velocity. A total of 79 transitions were removed due to this, and this was 0.04% of all 182 103 transitions detected in this data set.Age and sex were extracted from the population register, body height (stadiometer), body mass , socioeconomical status (self-reported years of education), and cognitive function test  were assessed using standardized procedures . Lower-eFear of falling was assessed by the question “Are you afraid of falling?” with 4 response options: never, occasionally, often, and constantly . In thisSD). Associations between variables were tested with Spearman rank correlation coefficients. Number, mean, and maximal angular velocity of STS transitions were categorized into tertiles group comparisons for 5×STS time and knee extension force normalized for body mass. Tertiles  were selected to maintain sufficient sample sizes in each group. Shapiro-Wilk normality test indicated that some of the variables were not normally distributed, and nonparametric statistical tests were therefore chosen. Sex and age group differences were analyzed with Mann–Whitney U (Wilcoxon rank-sum) test for categorical/dichotomous variables and Kruskal–Wallis test for continuous variables. Tertiles comparisons were performed using the Kruskal–Wallis multiple comparison test and the Dunn’s test (Holm-Bonferroni method) in pairwise comparisons. Sensitivity analyzes between sexes and age groups were performed between the variables and the self-reported questions . Statistical significance was set at p < .05 (2-tailed), and analyses were performed in the “R” statistical environment (version 4.1.1) (Results of STS transitions are reported as mean and standard deviation (n 4.1.1) .p < .001). The 85-year-old women showed 19.6% fewer STS transitions (p = .005) and 9.2% lower mean (p < .001) and 14.6% lower maximal angular velocity (p < .001) in the free-living environment compared to the 75-year-old women. The 85-year-old men showed 18.3% fewer STS transitions (p = .015) and 8.9% lower mean (p = .012), and 9.4% lower maximal (p = .042) angular velocity of the STS transitions compared to the 75-year-old men.Descriptive characteristics and free-living STS transitions of the participants are presented in r ranged from 0.18 to 0.39 all p < .001) and negatively associated with the 5×STS test . We also ran the correlation analyses for sexes and age-groups independently as a sensitivity analysis, and the correlations did not differ markedly between sexes and age groups.The Spearman rank correlation coefficients between variables are given in Tertiles group comparisons are presented in p < .001) and had 5.5% lower STS mean angular velocity (p < .001) and 8.9% maximal angular velocity (p < .001) in free-living conditions compared to individuals who reported no fear of falling p < .01) and had 6.9% lower STS mean angular velocity (p < .001) and 10.0% maximal angular velocity (p < .001) than individuals who reported no difficulties with stair negotiation p < .01) and had 5.8% lower STS mean velocity (p < .001) and 10.1% maximal angular velocity (p < .001) in free-living conditions compared to individuals who do not have lower extremity functional limitations Individuals who feared falling showed 15.8% fewer STS transitions  is congruent with previously published results. Bohannon (2015) stated in his review that the average number of STS transitions are at least 45 per day among most community-dwelling individuals . In the r = −0.12, p = .47) association between 5×STS test and free-living STS events in people with chronic low back pain  of mean or maximal angular velocity differed on lab-based lower-extremity performance from the other tertiles (T2 and T3), while no differences were found between tertiles T2 and T3. This could suggest that the mean and maximum angular velocities of the STS transitions only begin to decline when capacity is significantly impaired. Altogether, this could indicate that above a certain level of capacity, the intensity of the free-living STS transition does not increase linearly, which is well in line previously reported curvilinear relationship between knee extension force and 5×STS time . TherefoTo the authors’ best knowledge, the association between the knee extensor force and the angular velocity of STS transitions in a free-living environment has not been previously reported. Corrigan and Bohannon (2010) found a moderate correlation between knee extension force measured with a hand-held dynamometer and the duration of a single maximal STS transition performed in a laboratory, which is well in line with the correlation between knee extension force and maximal angular velocity of observed in the presents study. The association between 5×STS and STS transitions intensity was low. However, intensity quantified in this study (angular velocity) assessed only the STS phase, whereas 5×STS completion time also includes the stand-to-sit phase and any pauses between phases , which mr = −0.11, p = .009) associated with fear of falling  in free-living conditions  have previously reported that fear of falling is related to total daily physical activity  and the nditions . In addinditions . Explorinditions , but standitions . Given tThe decline in lower-extremity performance begins in middle-age, however, the decrements in physical capacity can be masked up to the age of 60–70 in submaximal activities such as walking . As STS Some limitations need to be kept in mind when interpreting the findings. Firstly, we demonstrated that identification and intensity quantification of STS transitions is possible based on thigh-worn accelerometer data in free-living conditions. However, validity  could only be examined for STS transitions identification , while tThe strength of this study can be considered a relatively large sample of community-dwelling participants. In addition, this study included multiple days (3–7 days) accelerometry recording, which is thought to be sufficient for assessing activity patterns . The strFree-living STS volume and intensity were positively associated with higher lower-extremity performance and negatively associated with lower extremity functional limitations, self-reported fear of falling, and stair negotiation difficulties. The number and mean and maximal velocity of STS transitions in free-living situations was lower with advancing age and differed between sexes. The intensity of STS transitions was more strongly related to lower-extremity performance than the number of STS transitions. Due to the strength-demanding nature of transitioning from sitting to standing, we hypothesize that the proposed free-living STS transition quantification may enable identifying those at risk of future limitations in daily activities.The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online.Supplementary data are available at glac071_suppl_Supplementary_AppendixClick here for additional data file.


The MHS was superior to ROC analysis  in detecting ICU-AW compared to MGS and MZS on day 10. The MHS correlated with the Medical Research Council sum score (MRC-SS) , the mRS  and BI  on day 100. Qualitative MUS analysis seems superior to quantitative greyscale analysis of muscle echogenicity for the detection of ICU-AW.Qualitative assessment by the Heckmatt scale (HS) and quantitative greyscale analysis of muscle echogenicity were compared for their value in detecting intensive care unit-acquired weakness (ICU-AW). We performed muscle ultrasound (MUS) of eight skeletal muscles on day 3 and day 10 after ICU admission. We calculated the global mean greyscale score (MGS), the global mean z-score (MZS) and the global mean Heckmatt score (MHS). Longitudinal outcome was defined by the modified Rankin scale (mRS) and Barthel index (BI) after 100 days. In total, 652 ultrasound pictures from 38 critically ill patients (18 with and 20 without ICU-AW) and 10 controls were analyzed. Patients with ICU-AW had a higher MHS on day 10 compared to patients without ICU-AW (2.6 (0.4) vs. 2.2 (0.4),  Muscle ultrasound (MUS) is a promising technique for the non-invasive detection of neuromuscular diseases, especially in critically ill patients who are at high risk of developing intensive care unit-acquired weakness (ICU-AW) ,2,3,4,5.Ultrasound data were derived from a prospective combined experimental and clinical study evaluating new approaches to diagnose ICU-AW in intensive care patients at two perioperative ICUs of a single university hospital. The study was approved by the local ethics committee of the University of Rostock in January 2016 (ethics identifier: AS 2016-0016). The study was registered as a clinical trial . The results of the experimental study part have already been published . WrittenThe Medical Research Council sum score (MRC-SS) was assessed on day 3 and day 10 by an ICU staff member (FK) who was blinded to the results of the MUS. Before clinical examination, analgo-sedation was interrupted, if present. Patients were eligible for assessment if five initial commands were carried out sufficiently . According to current recommendations, patients were diagnosed as ICU-AW positive (ICU-AW+) with an MRC-SS < 48 and ICU-AW negative (ICU-AW-) with an MRC-SS ≥ 48 on day 10 . Ultrasound examinations were performed by a consultant neurologist with 15 years of experience in MUS (UW) using a standardized in-house protocol see . The exaEchogenicity of each muscle was rated visually by using the four-grade HS as previously described : grade 1A trained research assistant (JosE) performed the greyscale analysis using the software ImageJ  and was blinded to the results of MUS and clinical examination. Prior to greyscale analysis using the histogram function, all images were converted into 8-bit greyscale images. A region of interest (ROI) was defined by marking the maximum visible cross-sectional area (CSA) of the muscle of interest excluding surrounding muscle fascia. Prominent intramuscular fascia and image artefacts were excluded from the ROI. As muscle CSA was shown to correlate with muscle strength , we defiThe greyscale level standard deviation (GSSD) within the ultrasound image was measured to determine muscle tissue homogeneity . The glot-test was used for normally distributed data, and the Mann–Whitney U-test to compare continuous or discrete variables. The Chi square test with Yates correction was used for categorical variables. In case of expected values of <5 in the 2 × 2 contingency table, Fisher‘s exact test was performed. The Pearson correlation coefficient was calculated to compare normally distributed variables; otherwise, we used the Spearman rank correlation coefficient. Receiver operating characteristics (ROC) with a 95% confidence interval were calculated to determine the discriminative ability of ME to detect ICU-AW. Statistical significance was indicated by p < 0.05. All statistical tests were two-sided. Since eight muscles were compared, a Bonferroni correction was employed with the significance level set at p ≤ 0.006 for the referring group comparisons.For statistical analysis, we used MS-Excel 2010  and IBM SPSS Statistics . Data are presented as sum (percent) or mean (standard deviation). Student’s We enrolled 51 postoperative critically ill patients and 10 healthy controls . Three pp values available in p = 0.002; ICU-AW− p = 0.02) and 10 .In total, 652 ultrasound images of 38 critically ill patients and 10 healthy controls were analyzed. Mean values and statistical data are summarized in p < 0.001) and 10 (1.1 (0.1), p < 0.001).Patients with and without ICU-AW had higher HS gradings compared to the control group on days 3 and day 10 . Furtherp values of group comparisons are available in p = 0.79; day 10: 72.1 (24.2) vs. 69.2 (27.5), p = 0.73), or in the MZS (day 3: 1.0 (0.8) vs. 0.9 (1.1), p = 0.73; day 10: 0.8 (0.9) vs. 0.7 (1.0), p = 0.73) between patients with and without ICU-AW.We found no differences in the greyscale values of any extremity muscle on day 3 or day 10 between ICU-AW+ and ICU-AW− patients (p = 0.07) on day 3 .We found no differences in most of the HS grading of single muscles between ICU-AW+ and ICU-AW− patients on day 3 . There wp = 0.003). Furthermore, using the MHS, we calculated an optimal cut-off value of 2.2 on day 10 with a sensitivity of 86% and a specificity of 60% to identify ICU-AW+ patients.To determine the diagnostic value of the parameter ME in MUS for the differentiation of ICU-AW+ and ICU-AW− patients, we performed a ROC analysis of the MGS, MZS and MHS for both assessments on days 3 and 10 . Only thp = 0.85), MZS (p = 0.34) or MHS (p = 0.05) in the population of ICU patients. On day 10, the MHS correlated with the corresponding MRC-SS (p = 0.004), but not the MGS (p = 0.90) or the MZS (p = 0.89). An increased MHS on day 10 was related to a decrease of the BI (p = 0.04) and an increase of the mRS (p = 0.007) on day 100 after study inclusion. In contrast, neither the MGS  nor the MZS  on day 10 correlated with functional outcome parameters.We found no correlation between the MRC-SS on day 3 and the corresponding MGS (Our study compared the utility of two different ultrasound methods to assess ME in critically ill patients at risk of ICU-AW. Specifically, we compared software-based greyscale image analysis and visual qualitative HS in this population. Our results indicate that both methods are suitable for the detection of increased ME in skeletal muscles of critically ill patients compared to healthy controls. However, in this study, only the summarized qualitative assessment of the echo intensity in multiple skeletal muscles represented by the MHS was able to identify patients with ICU-AW with good accuracy, and correlated with the severity of muscular weakness and functional impairment 100 days after study inclusion.Increased ME has been consistently reported in studies employing MUS in critically ill patients with neuromuscular dysfunction compared to ICU patients without polyneuromyopathy or healthy controls ,15,26,27A decline in muscle GSSD was associated with an increase of muscle homogeneity due to inflammation and muscle fiber breakdown . HoweverAlthough a possible association of increased ME values with a decline in muscle strength and function contributing to the development of an acquired muscle dysfunction has been hypothesized ,26, thisPillen and co-workers compared the quantitative and qualitative assessment of ME in 76 children with neuromuscular diseases . SensitiMuscle echogenicity was reported to correlate with age ,33 and fSome limitations of the present study must be mentioned. The study was designed as a pilot study without initial power calculation. Due to our strict exclusion criteria, only 51 patients were consecutively enrolled in this single-center study and data from just 38 patients were available to image analysis. In 10 patients, the MRC-SS could not be assessed on day 10. This represents a typical shortcoming in the daily clinical routine of simply using the MRC-SS to detect ICU-AW in critically ill patients, which underlines the diagnostic advantage of MUS in assessing patients independently from their ability to cooperate. The controls were not matched for age, so they were significantly younger than the ICU patients, possibly affecting ME. Despite careful conduct of the ultrasound examinations, changes in ME by unintentional slight tilting of the ultrasound probe cannot be fully ruled out. The visual assessment of ME is assumed to be more subjective, resulting in a moderate inter- and intra-observer agreement . TherefoOur findings support the use of a comprehensive muscular ultrasound protocol for the assessment of increased ME in critically ill patients to detect ICU-AW. In the present study, software-based greyscale analysis was not able to distinguish between patients with and without ICU-AW. We provide evidence that qualitative ultrasound imaging-based scores comprising several diagnostic criteria may be superior in detecting ME compared with a pure qualitative method. Future studies are warranted to verify the prognostic value of this valuable diagnostic tool if applied by less experienced, adequately trained investigators.


N-glycan of serum glycoproteins by mass spectrometry and lectin blotting. The results showed that bisecting GlcNAc structures of glycoproteins are significantly increased in PC patients’ sera. With Phaseolus vulgaris Erythroagglutinin (PHA-E) lectin that specifically recognizes bisecting GlcNAc N-glycans, the serum glycoproteins bearing bisecting GlcNAc in PC patients’ sera were pulled down and identified by nano-LC-MS/MS. Among them, ceruloplasmin (Cp) was screened out with a satisfied sensitivity and specificity in identifying PC from acute pancreatitis patients (AUC: 0.757) and normal healthy persons (AUC: 0.972), suggesting a close association between Cp and PC development and diagnosis. To prove that, the Cp expression in tumor tissues of PC patients was examined. The results showed that Cp was significantly upregulated in PC tissues compared to that in adjacent normal tissues. All these results suggested that PHA-E-positive Cp could be a potential PC-specific glycoprotein marker to distinguish PC patients from acute pancreatitis patients and normal persons.Pancreatic cancer (PC) remains one of the top 10 causes of cancer-related death in recent years. Approximately 80% of PC patients are diagnosed at the middle or advanced stage and miss the opportunity for surgery. The demand for early diagnostic methods and reliable biomarkers is increasing, although a number of tumor markers such as CA19-9 and CEA have already been utilized in clinics. In this study, we analyzed the alteration of  Pancreatic cancer (PC) is the seventh leading cause of cancer death in both sexes, with a mortality of 4.7% worldwide in 2020 . Its pooThe difficulty of early diagnosis is one of the main reasons for the high mortality of PC. Because no obvious symptoms are presented at the early stage of this disease, most patients are diagnosed at the middle or advanced stage of PC and miss the best time to achieve an optimal treatment . SurgicaN-GlcNAc modification on EGFR and small extracellular vesicles can diminish malignancy of breast cancer cells, which may contribute to the development of novel targets in breast cancer therapy [Disease-specific glycoproteins, which have glycoalterations in particular glycoproteins and can enhance the diagnostic sensitivity and specificity, attract much attention in biomarker developments in recent years . Glycosy therapy ,17. In t therapy ,19,20,21Ceruloplasmin (Cp) is known as a glycoprotein associated with malignancy in many cancers, including lung cancer and breast cancer . A previN-glycan in PC patients’ sera were analyzed and compared with those in the sera of acute pancreatitis (AP) patients and normal healthy individuals  by lectin blotting and MS. The results showed that bisecting GlcNAc structures of glycoproteins are significantly enhanced in the sera of PC patients. Serum glycoproteins with bisecting GlcNAc N-glycans were pulled down by PHA-E lectin in PC patients’ sera and identified by nano-LC-MS/MS. Among identified glycoproteins, we found that the expression of Cp and apolipoprotein E (Apo-E) in PC patients was significantly higher than those in AP patients and NC individuals after PHA-E pull-down. These data provide a theoretical basis for the diagnosis of PC patients and further exploration of the deep mechanism of Cp in PC progression.In this study, the expression of various types of The serum of AP and PC patients and NC individuals were obtained from Dalian Municipal Central Hospital affiliated to the Dalian Medical University, China. The clinical characteristics of these patients were presented in Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), PHA-E, Phaseolus vulgaris Leucoagglutinin (PHA-L), and Sambucus nigra agglutinin (SNA)  , were pun = 8, 300 μg) were dissolved in 30 μL of 80 mM ammonium bicarbonate containing 10 mM DL-dithiothreitol at 60 °C for 30 min, followed by adding 2.2 μL of 250 mM iodoacetamide and incubating at room temperature (RT) for 60 min in the dark. Then, the serum proteins were treated with 0.05 mg/mL trypsin  at 37 °C for 18 h. After heat-inactivation by trypsin at 95 °C for 5 min, the peptides were treated with 3 mU peptide N-glycanase F (PNGase F)  at 37 °C for 18 h and the generated whole-serum N-glycans were purified using the BlotGlyco® glycan purification kit according to the manufacturer’s protocol . Briefly, the glycans were captured by BlotGlyco® beads and methylesterified of sialic acids with 3-methyl-1-p-tolytriazene . Then, the glycans were labeled and released with an aminooxy-functionalized peptide reagent (aoWR), and the derivatized glycans were recovered from the resin by adding 50 μL distilled water. Finally, the excess reagents in the derivatized glycans were removed by using a cleanup column provided in the kit. The obtained solution containing the glycan derivatives was used for the following MS analysis.Pooled serum samples (The protein concentration of serum was determined by the bicinchoninic acid (BCA) protein assay kit (Takara Bio Inc.). For electrophoresis, serum proteins were incubated with SDS-PAGE sample buffer  at 100 °C for 5 min, loaded in the gel and separated in Tris-glycine SDS running buffer . After electrophoresis, the proteins were transferred onto the Immobilon-P PVDF membrane  at 80 mA for 1 h by using Tris-glycine SDS  transfer buffer with 20% methanol. To maximize the number of proteins retained on electroblotted PVDF membranes, a fixation method was performed as we reported recently [For lectin blotting, the fixed PVDF membranes were blocked with 5% BSA for 60 min and then washed with TBST solution  3 times (5 min each time), and finally incubated with biotinylated lectins at 4 °C overnight. The lectins were diluted in TBST at the work concentration of 0.13 μg/mL for AAL, 0.33 μg/mL for LCA, 0.1 μg/mL for PHA-E, 2 μg/mL for PHA-L, and 0.13 μg/mL for SNA. After being washed with TBST 3 times, the membranes were incubated with TBST diluted streptavidin-HRP for at RT 1 h. The bands were visualized using the ECL (enhanced chemiluminescence) Plus reagents (Beyotime). The protocol for immunoblotting was similar with that for lectin blotting, except for the antibody concentrations, membrane washing conditions, and the incubation time of secondary antibody. All primary antibodies were diluted in TBST at the ratio of 1:1000. After the incubation of primary antibodies, the membranes were washed with TBST for four times (10 min each time), incubated with the secondary antibody at RT for 2 h, and finally visualized by ECL Plus reagents.For each lectin blotting and immunoblotting, chemiluminescent signals were recorded using a ChemiDoc MP imaging system , and Image J software was used to quantify the protein bands. The total protein signal in Coomassie brilliant blue (CBB) staining of each sample was chosen as a standard control. The relative intensity was calculated by dividing the signal of each line in lectin blotting or a band in Western blotting with the total protein signal of corresponding line in CBB staining.g for 20 min at 4 °C. After being washed with 1 mL acetone in ice for 15 min and centrifuged again, the proteins were resolved in 2 M urea and their concentrations were determined using the BCA protein assay kit. For lectin pull-down, 350 μg proteins were incubated with biotin-PHA-E lectin at 4 °C overnight. Then, the streptavidin-agarose was added to the mixture and incubated at 4 °C for 3 h, and the captured proteins were analyzed with 10% SDS-PAGE. The bands of proteins that were differently expressed among NC, AP and PC groups were finally extracted from the SDS gel and identified by LC-MS/MS.To improve the pull-down efficiency of the proteins that contain bisecting GlcNAc by PHA-E lectin, we first removed the albumin, which is the most abundant serum protein according to previous reports . Total sThe gel bands were cut into smaller pieces and transferred into microtubes. After washed with Milli-Q water (Millipore), the gels were destained with 50 mM ammonium bicarbonate dissolved in acetonitrile (Sigma-Aldrich). After completely destaining and residual detergents removing, the samples were dehydrated in acetonitrile. When the gel pieces became opaque white, they were reduced and alkylated, respectively. Next, the gel pieces were digested for 16 h by trypsin (Sigma-Aldrich) at 37 °C. The peptide mixtures were collected and transferred into new microtubes for further analysis by Michrom AdvanceTM nano/cap LC-Q-TOF MS .v/v) formic acid in water, while mobile phase B consisted of 0.1% (v/v) formic acid in acetonitrile, and gradient conditions were as follows: 5 to 40% B in 80 min, up to 80% B in 4 min, maintaining this concentration for 10 min. Eluted peptides were directly introduced into a CaptiveSpray Ionization (CSI)-Q-TOF MS  for analysis. Dry temperature to 165 °C and capillary voltage to 1500 V. MS1 spectra were acquired from 50 to 2200 m/z at about 20,000 resolution (for m/z 445.1200). For each spectrum, the five most intense ions were subjected to collision-induced dissociation (CID) fragmentation, followed by MS2 analyzer.From the peptide mixtures, 5 μL solution was initially applied to a 2 cm long  trap column packed with 5 μm, 200 A Magic C18 AQ matrix  followed by separation on a 15 cm long column that was packed with 3 μm, 200 A Magic C18 AQ. Samples were loaded onto the trap column at 10,000 nL/min, and chromatographic separation occurred at 200 nL/min. Mobile phase A consisted of 0.1% , and Proteinscape 3.0  to identify peptides. Peak lists of raw files were generated by using Mascot Daemon  and searched against the Swiss-Prot or NCBI protein databases by using the search program Mascot server.2O2) solution and blocked with 5% fetal goat serum. Next, the sections were incubated with rabbit polyclonal antibody, anti-Cp  at 4 °C overnight. After washing with PBS, sections were incubated with a biotinylated secondary antibody at 37 °C for 1 h. Subsequently, sections were treated with diaminobenzidine , stained with hematoxylin and dehydrated using gradient alcohol. Finally, an Aperio Scanscope XT  was used to digitally scan the slides. IHC score counting was performed by ImmunoRatio  to prevent manual or interobserver bias. It was applied to calculate the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (DAB and hematoxylin) and adaptive thresholding for nuclear area segmentation. Every digital image was quantitatively analyzed in three independent areas.Formalin-fixed and paraffin-embedded tissue section samples  were prepared. Then, these sections were deparaffinized with xylene, rehydrated by alcohol gradient, followed by antigen retrieval by sodium citrate. The sections were incubated in 3% hydrogen peroxide , GEPIA  and BBCancer . Their difference between PC and adjacent normal tissues was compared by paired t-test.The expression of Cp and GnT III in PC and adjacent normal tissues was obtained from databases of GSE28735 database  from 8 or 12 individuals of one group, or from the triple tests of each group. Graphpad Prism 6.01 software  was used for statistical analysis and one-way ANOVA was selected to determine the significance of differences among the examined groups. N-glycan patterns among NC persons and AP and PC patients, the serum glycoproteins from 24 individuals, comprising 8 NC persons, 8 AP patients, and 8 PC patients  and AP patients (p < 0.05).To confirm the glycosylation alterations in the serum proteins of PC patients, ed by MS a. All glt charts b, along an types c. In totp < 0.01).Since glycans are difficult to be developed as biomarkers alone, we analyzed the serum glycoproteins containing bisecting glycans among three groups by pulling down with biotinylated PHA-E lectin and streptavidin agarose beads. The extracted PHA-E binding proteins were analyzed by SDS-PAGE a. The dip < 0.01), but also between AP and PC samples (p < 0.05)  . The AUC < 0.05) e. All thTo confirm the role of Cp in PC tumorigenesis, we detected the level of Cp in tumor tissues and adjacent noncancerous tissues from 3 PC patients by IHC staining. The results showed that the expression of Cp in tumor tissues was significantly higher than that in paired adjacent noncancerous tissues from the same patient a. To reaN-glycosylation of serum proteins were dramatically changed in both PC and AP patients [PC is a highly malignant solid tumor and usually demonstrates aggressive metastasis . Its maipatients . TherefoN-glycans of pancreatic ribonuclease also increased in PC tissues [N-glycans via the β1,4-linkage to make a bisecting GlcNAc structure in N-glycan biosynthetic pathways. The addition of a bisecting GlcNAc by GnT-III to target proteins typically inhibits the action of other glycosyltransferases such as GnT-IV and GnT-V. Therefore, GnT-III can act as a regulator of other glycosyltransferases [In this study, the alteration of glycans in the sera of NC individuals and AP and PC patients were investigated by MS and Lectin blotting. The results of alterations of fucosylation, sialylation and high mannose were consistent with the previous studies on PC cell lines  and the  tissues . GnT-IIIsferases . Aberransferases ,17,45. Hsferases . TherefoN-glycan was only detected in PC patients’ sera (peak 40), which indicated that this glycan may become a potential biomarker for the diagnosis of PC. In fact, Nouso et al. detected the expression of glycans in the sera of 92 PC patients and 243 NC through glycomics analysis and found that 15 glycans including this glycan were increased in PC, and 10 glycans were significantly upregulated in distant metastasis cases. In the diagnosis respect of pancreatic cancer, this glycan showed the AUC = 0.723 [In  = 0.723 .Besides bisecting GlcNAc glycans, proteins were also selected as biomarker candidates in this study based upon the following characteristics: (1) Protein functions were involved in cancer progression; (2) the MWs of protein candidates are similar to the sizes of the corresponding bands that we cut and analyzed; (3) the glycan structures of the proteins were not previously reported in PC; (4) commercially available antibodies exist for further immunoassays. Among all the PHA-E pulled-down proteins, Cp and Apo-E were chosen for the further analysis of their expression levels in PC, AP, and NC groups. The results indicated that the protein levels of Cp and Apo-E were increased in both AP and PC patients, but there was no obvious difference between PC and AP patients. However, statistical significance was observed in the Cp expression between the AP and PC samples after the proteins were pulled down by PHA-E lectin. Thus, the measurements of PHA-E-positive Cp by Western blotting and lectin blotting would be a more favorable indicator for PC in terms of glycoalteration. In the case of Apo-E, no significant difference was found between the AP and PC samples either in serum or PHA-E captured serum. A previous report showed that the elevation of Apo-E in pancreatic cancer is likely related with the obstructive jaundice rather than Apo-E acting as a direct marker of pancreatic malignancy . In addiIt is well known that most candidate biomarkers for cancer are associated with cellular processes of tumor development and malignant progression. Cp is a member of the multicopper oxidase family of enzymes. Previous reports showed that Cp is not only synthesized in the liver, but also produced by cancer cells . Tumor cIn summary, our findings showed that PHA-E-positive Cp was overexpressed in PC patients’ sera and tumor tissues. Using PHA-E and anti-Cp antibody as probes, PC could be distinguished from NC and AP patients, especially from NC individuals. Therefore, PHA-E-positive Cp is a potential biomarker for the diagnosis of PC, which needs to be validated and researched further for clinical use.


Long-term excessive intake of high-calorie foods might lead to cognitive impairments and overweight or obesity. The current study aimed to examine the effects of high-calorie foods on the behavioral and neurological correlates of food-related conflict control ability. A food-related Stroop task, which asked the participants to respond to the food images and ignore the calorie information, were employed. A total of 61 individuals were recruited and who completed the food-related Stroop task with event-related potentials (ERPs). Participants exhibited a slower reaction time and lower accuracy in high-calorie food stimuli than that in low-calorie food stimuli. The ERP results exhibited a reduction in N2 amplitudes when responding to high-calorie food stimuli compared to when responding to low-calorie food stimuli. In addition, time-frequency analysis revealed that theta power induced by low-calorie food stimuli was significantly greater than that of high-calorie food stimuli. The findings indicated that high-calorie foods impair food-related conflict control. The present study expands on the previous studies of the neural correlates of food cues and provides new insights into the processing and resolving of conflicting information for eating behavior and weight control. We live in an environment filled with various food temptations at our fingertips and we are constantly struggling between maintaining a healthy eating style and giving in to immediate food temptations. The excessive intake of high-calorie foods is considered a contributing factor to obesity, while resisting high-calorie foods is considered to require successful executive function ,2,3,4. BConflict control refers to the information processing capability of the brain to monitor and respond to the emergence of conflicts and is considered a core component of the brain’s executive function . PreviouAdding to behavior studies, prior neurophysiology studies have also investigated the relationships between inhibition control and food-cues. Although most of the previous studies have shown that high-calorie foods have a wide range of effects on P2, N2, P3, N450, and late positive potential (LPP) amplitudes, they have revealed mixed findings. Asmaro and his colleagues compared the neural-difference induced by high-calorie foods  and neutral objects  during discrimination tasks using event-related potentials , and found that high-calorie foods elicited significant enhancement of P2 and LPP amplitudes. The findings indicated that participants exhibited attentional bias by allocating more attentional resources to high calorie food stimuli compared to general stimuli . When asTheta power has also been a neural marker of focus for previous studies when discussing conflict control ability regarding food-related stimuli. Theta power of the medial frontal cortex is sensitive to perception conflicts and reaction conflicts. Theta band activities are usually involved in conflict processing, especially in the neural network that connects reaction conflicts ,28. In aUp to now, there have been few EEG studies that have utilized a food-related Stroop task to investigate the effects of high-calorie foods on conflict control. To add to previous studies, the present study examined the influence of high-calorie food cues on the neural correlates of conflict control. Based on previous studies, the neural correlates discussed in the present study are N2, P3, and theta power and we hypothesized that high-calorie foods would impair conflict control ability, which would be reflected by a slower reaction time, lower accuracy, a reduction in N2 amplitudes and theta power, as well as an increase in P3 amplitudes when reacting to high-calorie foods compared to when reacting to low-calorie foods during the food-related Stroop task.n = 61; 29 females; Mage = 20.25 years, SDage = 1.96; MBMI = 21.75 kg/m2, SDBMI = 2.98) were recruited from Chongqing Normal University, Chongqing. Before participating in the study, all participants were required to abstain from substances (including tea and coffee) and medications that could potentially affect their concentration during the study, and were required to disclose any history of psychological disorders. They were also required to have normal or corrected-to-normal vision. The participants were given the chance to ask any questions about their participation before consenting to the study. The present study was approved by the Southwest University Ethics Committee.Participants  × 2 (congruence: congruent and incongruent) repeated measures ANOVA was conducted on the reaction time (RT) and accuracy (ACC). All analyses were conducted by SPSS 25.0. The EEG data were recorded from 64 scalp sites using tin electrodes mounted in an elastic cap , with the reference electrode placed on the fronto-central aspect and a ground electrode on the medial frontal aspect. All inter-electrode impedance was maintained below 5 KΩ.EEG data preprocessing was performed via EEGLAB, an open-source toolbox available on the Matlab software. Individual and grand ERP averages were created for the food-related Stroop task. We first down sampled the data from 1000 Hz to 256 Hz and performed high-pass filtering at 0.1 Hz and low-pass filtering at 45 Hz. The mean values of the left and right mastoids were selected as the re-reference. Data were epoched from 200 ms prior to stimuli onset to 1000 ms after the onset, and were baseline corrected to the pre-stimuli interval. Trials with large fluctuations in amplitudes were removed before the independent component analysis (ICA). The components, including EOG artifacts (ocular movements and eye blinks) and head movement, were then removed from the results of the ICA. Based on the topographical distribution of the grand-average ERP activities, the ERP components and their time window were as follows: N2 (250–360 ms), and P3 (360–400 ms). Based on previous studies, the following electrode sites were selected, Fz, FCz, Cz, CPz, and Pz. Two 2  × 2 (congruence: congruent and incongruent) × 5  repeated measures ANOVAs were conducted on the mean amplitudes of N2 and P3.F  at each point  of the time-frequency plane extending from −200 ms to 1000 ms in the time domain, and from 1 Hz to 30 Hz (in step of 1 Hz) in the frequency domain, for each single trial. A baseline correction was applied at the subject level using the pre-stimulus interval (pre-stimulus −200 to 0 ms) to calculate the change of power according to the formula:, and R(f) is the averaged power spectral density of the signal enclosed within the pre-stimulus reference interval (−200 to 0 ms before the onset of the stimulation) for each estimated frequency f. Brain rhythmic activity of the theta  was selected in the current analysis. A 2  × 2 (congruence: congruent and incongruent) × 5  repeated measures ANOVA was conducted on the value of theta.The time-frequency analysis for the EEG data, employed a windowed Fourier transform (WFT) with a fixed 250 ms width Hanning window. The WFT yielded a complex time-frequency spectral estimate p-values were computed for deviation in all analyses. Post-hoc t-tests were conducted with Bonferroni correction for multiple pairwise comparisons.All analyses were conducted via SPSS 25.0. Based on the Greenhouse–Geisser method, F  = 39.30, p < 0.001, partial η2 = 0.40, RT to high-calorie foods was significantly greater than that to low-calorie foods. The results on RT also showed a main effect of congruence, F  = 39.30, p = 0.001, partial η2 = 0.16, RT in incongruent trials was significantly greater than that in congruent trials.The results on RT A showed F  = 19.48, p < 0.001, partial η2 = 0.25, ACC of high-calorie foods was significantly lower than that of low-calorie foods. The results on ACC also showed a main effect of congruence, F  = 5.01, p = 0.03, partial η2 = 0.08, ACC in congruent trials was significantly greater than that in incongruent trials.The results on ACC B showed Grand average ERPs for N2 and P3 at Fz and topography plots are shown in N2F  = 13.55, p < 0.001, partial η2 = 0.18. The simple effect analysis showed that N2 amplitudes of low-calorie foods were significantly greater than those of high-calorie foods at Fz, F  = 4.97, p = 0.03, partial η2 = 0.08. The results also showed an interaction of congruence and electrode site, F  = 3.18, p = 0.048, partial η2 = 0.05. The simple effect analysis showed that N2 amplitudes in incongruent trials were significantly greater than those in congruent trials at Fz , FCz , and Cz .The results on N2 amplitudes showed an interaction of food and electrode site, P3F  = 24.55, p < 0.001, partial η2 = 0.29, P3 amplitudes in congruent trials were significantly greater than those in incongruent trials.The results on P3 amplitudes showed a main effect of congruence, ThetaF  = 8.25, p = 0.006, partial η2 = 0.12, theta power of low-calorie foods (0.77) was significantly greater than that of high-calorie foods (0.59). The results also showed an interaction of congruence and electrode site, F  = 9.46, p = 0.001, partial η2 = 0.14. The simple effect analysis showed that theta power in incongruent trials was significantly greater than that in congruent trials at Pz, F  = 4.15, p = 0.046, partial η2 = 0.07.The results on theta showed a main effect of food, The present study used an originally modified food-related Stroop task to explore the effects of high-calorie foods on conflict control and the underlying neurophysiological responses. The hypothesis was partially confirmed by our original method of examination. The results showed that the RT to high-calorie food cues was greater than that to low-calorie food cues, whereas the ACC of high-calorie food cues was significantly lower than that of low-calorie food cues. We also found that high-calorie foods induced lower N2 amplitudes and theta power compared to low-calorie foods. No significant difference in P3 amplitudes were found between high-calorie and low-calorie food cues.As our modified task elicited data from both the mental processing of food images combined with calorie information, as well as motor response performance, the current results support previous findings that high-calorie foods impair cognitive performance . Adding The present study showed a reduction in N2 amplitudes in high-calorie foods compared to low-calorie foods. Depending on the stimuli and tasks displayed, N2 amplitudes reflect sensory processing, response inhibition, and conflict monitoring ,40. FolsThe result of decreased theta power in high-calorie foods was consistent with the result of reduced N2 amplitudes in high-calorie foods. Frontal midline theta is located in the anterior cingulate cortex (ACC) and the auxiliary frontal motor area of the medial frontal cortex (MFC)  and is pHowever, we did not find a difference in P3 amplitudes between high-calorie foods and low-calorie foods. P3 reflects various cognitive processes including working memory and attention allocation . A previThe present study has several limitations. First, the participants’ food cravings were not measured. The relationship between food craving and food-related conflict control was therefore not explored in the current study. Future research could measure the state of the food craving  before and after the food-related Stroop task. Second, the current study focused on the temporal measures of brain activity of the EEG data, hence the lack of spatial measures of brain activity. Future studies could explore both the temporal and spatial neural indices in a food-related Stroop task by collecting both EEG and fMRI data. Third, general stimuli  were not adopted in the current study, and the influence of other characteristics such as the stimuli colors or nutrient density on the experimental findings was not considered in the present study, which might be worth exploring in future studies.In conclusion, this study uses an originally modified food-specific Stroop task and EEG to complement previous literatures. The current original version of the food-related Stroop task may be a promising instrument to be implicated in future brain imaging studies to further demonstrate the nuances of various types of foods on cognitive processing. Furthermore, the task could be adopted to explore the underpinnings of the relationship between high-calorie foods and conflict control, including its behavioral consequences. The study has shown that high-calorie foods impair food-related conflict control ability, expanding the studies of the neural mechanism of food cues while providing new insights into the processing and solving of conflicting information for healthier eating behavior and weight control.